Distribution of 5 alpha-reductase in the epididymis of the tammar wallaby (Macropus eugenii) and dependence of the epididymis on systemic testosterone and luminal fluids from the testis

The activity of 5 alpha-reductase was much higher in the caput and corpus epididymidis than in the cauda epididymidis. Orchidectomy caused a reduction in 5 alpha-reductase activity in the caput and corpus epididymidis, and regression of the epithelium and reduction in mass of all regions of the epididymis. Subsequent testosterone therapy caused a substantial increase in amount of epithelium and overall mass of the cauda epididymidis but showed little or no increase in any of the responses measured in the caput and corpus epididymidis. We concluded that the caput and corpus epididymidis of the tammar respond to factors other than testosterone, probably some constituent in the luminal fluid, and therefore are homologous with the initial segments of the epididymis in eutherians.     

Subcellular distribution of steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during sexual maturation

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and ovarian control of progestin-metabolizing enzymes in various rat hypothalamic regions

The three principal hypothalamic progesterone metabolizing enzyme activities, namely the progesterone 5 alpha-reductase and 5 alpha-dihydroprogesterone NADH- and NADPH-linked 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities, were examined in discrete rat hypothalamic subsections throughout the estrous cycle and from ovariectomized rats treated with estradiol benzoate or vehicle. The regions studied included the median eminence, the medial preoptic area and the ventromedial and arcuate nuclei. The enzyme assays were performed using radiolabeled steroid substrates and reverse isotopic dilution analysis. While all four hypothalamic regions obtained from intact cycling animals possessed substantial amounts of these three enzyme activities, the median eminence generally had the highest activity levels (2- to 4-fold greater) except during estrus. The other three regions usually had comparable levels. No significant fluctuations were observed in any enzyme activity over the estrous cycle. After ovariectomy, there was a significant decrease (approximately 35%) in the level of the NADPH-linked 3 alpha-HSOR activity in the median eminence compared to the level observed in intact cycling animals, suggesting ovarian control. Estrogen treatment for 3 days did not restore this enzyme level to that observed in intact animals. The NADPH-linked 3 alpha-HSOR activity from the other three hypothalamic regions, as well as the NADH-linked 3 alpha-HSOR and the 5 alpha-reductase activities from all four brain regions, did not change significantly after ovariectomy. These results indicate that the median eminence possesses an increased capacity for progesterone metabolism relative to the other hypothalamic regions tested, and that the NADPH-linked 3 alpha-HSOR activity in this region may be under ovarian control.     

Potent inhibition of the hypothalamic progesterone 5 alpha-reductase by a 5 alpha-dihydroprogesterone analog

The ability of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit the progesterone 5 alpha-reductase and the two 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADH- and NADPH-linked) from female rat hypothalamus has been studied. Dose response experiments indicate that AMPD is a potent antagonist of hypothalamic progesterone 5 alpha-reduction but is an ineffective inhibitor of the NADPH- and NADH-linked 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. Kinetic analyses of the interaction of AMPD with the progesterone 5 alpha-reductase show that it is a competitive inhibitor versus progesterone (Ki(slope) = 6.2 +/- 0.5 nM; apparent Km (progesterone) = 130 +/- 12 nM) and an uncompetitive inhibitor versus NADPH (Ki(intercept) = 11.8 +/- 0.8 nM). These inhibition patterns are consistent with the concept that NADPH binding precedes that of either AMPD or progesterone. The inhibition of the progesterone 5 alpha-reductase by AMPD does not appear irreversible since preincubation of the enzymatic activity (at 37 degrees C) with inhibitor and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. Furthermore, this inhibition can be easily removed via dilution, even following a 60-min preincubation with AMPD and NADPH. It is postulated that the specific and powerful inhibition of the progesterone 5 alpha-reductase by AMPD may be due to this compound functioning as a transition state analog. This inhibitor should prove valuable in studying the characteristics of the progesterone 5 alpha-reductase and the function of hypothalamic progestin metabolism.     

Changes in lipase and phosphatase activities of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis, freed from their cytoplasmic droplets and acrosomes, were found to have a lower lipid content and to incorporate [14C]glucose into their glycerides and glycerophosphatides at a lower rate than spermatozoa from the caput epididymidis. Against the background of the activities of some glycolytic enzymes which remained constant the activity of alkaline phosphatase decreased in spermatozoa migrating through the epididymis, whereas the activity of monoglyceride lipase increased. The corresponding enzyme activities of non-flagellate germ cells of the testis were measured for comparison. The triglyceride lipase of non-flagellate germ cells and of spermatozoa from both caput and cauda epididymidis was activated by cyclic 3':5'-AMP.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.     

Effects of castration, 5 alpha-dihydrotestosterone and cyproterone acetate on enzyme activity in the mouse epididymis

The influence of castration, androgen replacement therapy and cyproterone acetate on the activity of beta-glucuronidase, acid and alkaline phosphatases and glucose-6-phosphate dehydrogenase was studied in the caput, corpus and cauda epididymidis of the mouse. The results add further evidence that the epididymis is not uniform but has regional differences in activity. Thus beta-glucuronidase was found to be androgen-dependent only in the cauda epididymidis, whereas glucose-6-phosphate dehydrogenase was under androgenic control in the caput epididymidis. The response of alkaline and acid phosphatases to castration and to androgen replacement was different in different segments.     

Effect of allylisopropylacetamide and Sedormid on enzymes of steroid metabolism in rat liver

Female rats, treated with allylisopropylacetamide (AIA) showed a marked decrease of hepatic NADH-5 alpha-reductase, NADPH-5 alpha-reductase, NAD+- and NADP+-3 alpha-hydroxysteroid dehydrogenase activities and an increase of the activity of NADH- and NADPH-5 beta-reductase and NAD+ and NADP+-3 beta-hydroxysteroid dehydrogenase. Administration of Sedormid decreased the activities of 5 alpha-reductases and 3 alpha-hydroxysteroid dehydrogenases (substrate, 5 alpha-dihydrotestosterone) and increased the activity of NADH-5 beta-reductase, whereas no effect was seen on NADPH-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase.     

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5alpha-reductase and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5alpha-reduced progesterone metabolites such as 5alpha-dihydroprogesterone (5alphaP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaHP) have the opposite (anti-cancer-like) effects. The 5alpha-reductase inhibitor dutasteride has been shown to inhibit 5alpha-reduction of testosterone to 5alpha-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5alpha-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10(-6)M) inhibited progesterone conversion to 5alpha-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5alpha-reductase activity and to altered levels of expression of 5alpha-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5alpha-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5alphaP, providing proof-of-principle that the effects were due not to progesterone but to the 5alpha-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5alpha-reductase inhibitor and that such inhibition may be beneficial in breast cancer.     

Pituitary progestin-metabolizing enzyme activities in the aged female rat.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) enzymic activities (NADH-linked and NADPH-linked) were measured in anterior pituitaries (AP) from aged female rats during three stages of reproductive senescence (constant estrus: CE; repeated pseudopregnancies: PSP; and anestrus: AN). To assess ovarian influence on these enzymes during these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3 alpha-HSOR levels did not differ among the three stages. All three enzyme levels were elevated 2- to 5-fold as compared to the corresponding enzyme levels from young cycling rats. After ovariectomy (10 days), 5 alpha-reductase activity in PSP and AN rats was elevated 3- to 4-fold relative to mean levels in intact PSP and AN rats. Ovariectomy had no effect on 5 alpha-reductase levels in CE rats. Under similar conditions, young cycling rats exhibit a 10-12-fold increase. Treatment of ovariectomized PSP and AN rats for 3 days with estradiol benzoate (10 micrograms/day) restored 5 alpha-reductase levels. Ovariectomy had no effect on the NADPH-linked 3 alpha-HSOR levels in CE, PSP or AN animals which is similar to that observed with young rats. Ovariectomy also had no effect on the NADH-linked 3 alpha-HSOR levels except for the CE group. The ovariectomized CE rats exhibited reduced pituitary NADH-linked 3 alpha-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3 alpha-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5 alpha- and 3 alpha-reduced products may be one means by which the effectiveness of progesterone is reduced during aging. The results also suggest an altered ovarian role in the regulation of these enzymes.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

The histochemical localization of prostaglandin synthetase activity in reproductive tract of the male rat.

PG synthetase activity was assessed histochemically in the reproductive tract of male rats. Moderate activity was observed in tails of spermatozoa within the corpus and cauda epididymidis but there was no activity in the caput epididymidis or the seminiferous tubules. The sperm tail activity was maximal for cells within the vas deferens. PG synthetase activity was also observed in individual adipose cells adhering to the testicular capsule, epididymis and vas deferens, and in isolated interstitial cells of the testis and the caput, corpus and cauda epididymidis. Specific cells in the capsules of the testes, epididymis and vas deferens also produced PGs. The activity observed in the interstitial cells of the testis and the caput epididymidis was less than that for the other tissues in terms of the proportion of possible cells. The demonstration of PG synthetase activity paralleled to known loss of arachidonic acid from the phospholipids of the spermatozoa as they pass through the male tract. Endogenous substrate was not limiting in the assay system, even in the testis and caput epididymidis where PG synthesis was not normally observed, indicating that a PG synthesis inhibitor may be present in these two tissues. PG synthetase activity within teased seminiferous tubules was markedly increased by physical trauma. Indomethacin diminished but did not eliminate synthesis.     

NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.     

Androgenic actions of 5 alpha-androstane-3 alpha,17 beta-diol in rat submandibular gland

To evaluate the action of 5 alpha-androstane-3 alpha,17 beta-diol(3 alpha-diol) in rat submandibular gland, 5 alpha-reductase, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) and oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDO) activities, and trypsin-like protease activities, were assayed in control, castrated and 3 alpha-diol injected rats. 3 alpha-Diol (1 mg/kg) was injected subcutaneously in castrated male rats daily for 7 days. A 47% decrease of 5 alpha-reductase activity in the nuclei and a 30% decrease of 3 alpha-HSD(O) activities in the cytosol were shown after castration. 3 alpha-Diol restored the 5 alpha-reductase and 3 alpha-HSD(O) activities to 82 and 140% of the control submandibular gland, respectively. 3 alpha-Diol raised the trypsin-like protease activity to near control values in the submandibular gland of castrated rats. Morphological observations also revealed a distinct effect of 3 alpha-diol on the number of granules of granular duct cells. It is concluded that 3 alpha-diol has an androgenic action in the rat submandibular gland. It stimulates the 3 alpha-HSD(O). The 3 alpha-HSD(O) in its turn may be responsible for DHT accumulation in the cells.     

beta-N-acetylglucosaminidase in the reproductive organs and seminal plasma of the bull

The highest specific activity of beta-N-acetylglucosaminidase (beta-NAG) was found in the different parts of the epididymis, where the activity seemed to be partly in secretory and partly in non-secretory, tissue-bound form. Epididymal spermatozoa also contained moderate beta-NAG activity. The beta-NAG was separated by chromatofocussing and anion exchange chromatography with HPLC into multiple forms with distinct pI values (8.0-4.0). The cauda epididymidis, ampulla and the seminal vesicles formed the major secretory sources of the high beta-NAG activity in bull seminal plasma. The major secretory forms of beta-NAG in caput and cauda epididymidis showed distinct elution profiles. In the fractionation with gel filtration on Sepharose 6B, the beta-NAG activities derived from bull testis and caput epididymidis had smaller molecular weights than did the secretory enzymes in seminal plasma, seminal vesicle secretion and cauda epididymidis. Maximum activity of all beta-NAG isoenzymes was observed at pH 5.0. They were almost totally inactivated at 60 degrees C and about 75-80% of the activity was lost at 55 degrees C. All the isoenzymes were strongly inhibited by thiol reagents but not with other metal ions and chelating agents. Histochemical studies showed a strong granular (lysosomal) reaction for beta-NAG in basal cells and basal parts of the principal cells in all but the initial segment of the epididymis. An apical (secretory) reaction was prominent in the distal caput and corpus as well as in distal cauda. After the distal caput the luminal sperm mass became increasingly mixed with a beta-NAG-positive material. The epithelial cells of the ampulla and seminal vesicle displayed a moderate apical (secretory) reaction.     

Changes in surface ATPase of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis were found to have a lower activity of the surface ATPase than the spermatozoa from the caput region. The enzyme from spermatozoa of both regions had the same Michaelis constant (Km) for ATP of 5 X 10(-4) M. It was partly inhibited by ouabain and fluoride, but strongly inhibited by Cu2+, Zn2+,p-chloromercuribenzoate, 8-anilino-1-naphthalenesulphonate Triton X-100, Lubrol-PX, urea, guanidine hydrochloride, sodium dodecyl sulphate and glycerylphosphorylcholine. The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride but less sensitive to inhibition by Cu2+ than that of the cells form the caput region. The Arrhenius plot of the temperature dependence of enzymatic activity varied for the cells from the caput and cauda epididymidis. The differences in the enzyme properties of spermatozoa from the two regions of the epididymis suggested that the decline in the activity during epididymal maturation may reflect changes in the lipids and sulphydryl groups of the sperm membrane.     

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.     

The lipid content of epididymal spermatozoa of Rattus norvegicus.

1. Seven major lipids of rat spermatozoa from the caput, corpus and cauda epididymidis were separated and quantitated by TLC. 2. Spermatozoa from the caput epididymidis had a significantly greater (P less than 0.05) content of phospholipid, cholesterol, cholesterol ester and free fatty acid than those from the cauda epididymidis. 3. Spermatozoa from the corpus epididymidis had a significantly greater (P less than 0.05) content of monoglyceride than those from the caput epididymidis and a greater content of phospholipid, cholesterol, free fatty acids and monoglyceride than those from the cauda epididymidis.     

A determinant of Mr 34,000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture

A murine monoclonal antibody raised against hamster cauda epididymal spermatozoa was shown to recognize an Mr 34,000 component of epididymal epithelium. Antigen was localized by immunocytochemistry on the surface and in the apical cytoplasm of principal cells in the proximal corpus epididymidis but not in the caput or initial segment regions. Spermatozoa from the corpus epididymidis expressed antigen on their post-acrosomal plasma membrane and annulus. Epididymal principal cells from the proximal corpus region when cultured in vitro bound antibody on their apical surface for at least 5 days. Spermatozoa from the caput epididymidis co-cultured with epithelium expressed antigen after incubation for 8 and 24 h. These results suggest that a surface change to epididymal spermatozoa during maturation in vivo may also be elicited during in-vitro culture.